The stratum corneum of terrestrial mammals is now accepted to be a heterogeneous structure of protein-enriched intercellular domains. This "two-compartment model" explains many phenomena of importance for cutaneous barrier function. The intercellular lipids of the stratum corneum appear to derive from the secretion of a distinctive organelle, the epidermal lamellar body, which possesses a distinctive profile of lipids, and a select group of hydrolytic enzymes. With the establishment of a lifted-culture model, where human keratinocytes elaborate lamellar bodies when grown at an air/medium interface, and a new isolation technique for lamellar bodies, we will now study the lamellar body secretory system. Specifically, we will use pharmacologic agents in the lifted culture system, to separately assess the contents, site of synthesis, assembly, kinetics, translocation, targeting, and exocytosis of lamellar bodies. Finally, the intercellular transformation of secreted lamellar body contents into hydrophobic lamellae in the stratum corneum interstices will be studied using a new lipid-retaining, rapid-freeze protocol. A second major goal of this proposal is to determine how environmental factors and dietary fatty acids influence barrier structure, function, and composition. Third, we well determine how barrier requirements regulate epidermal lipid biosynthesis, and which lipids and enzymes are specifically affected. For these studies we will use a series of short-term (SDS, acetone-treatment) and long-term (essential fatty acid deficiency, retinoid-treatment) models of barrier disruption in hairless mice. Fourth, we will study water flux as a regulatory signal to lipid biosynthesis. Finally, the ability of specific lipids to influence barrier function will be studied in model systems that should also shed light on the molecular basis for stratum corneum permeability. These studies should provide new strategies for the regulation of cutaneous barrier function that could result in novel forms of therapy for burn wounds, and improved delivery of topical drugs.